Identifying Genetic Variation for Alcohol Dependence

Researchers are using various strategies to identify the genes that may be associated with alcoholism. The initial efforts primarily relied on candidate gene and linkage studies; more recently, however, modern advances in genotyping have resulted in widespread use of genome-wide association studies for alcohol dependence. The key findings of the earlier studies were that variations (i.e., polymorphisms) in the DNA sequences of the genes encoding alcohol dehydrogenase 1B (i.e., the ADH1B gene), aldehyde dehydrogenase 2 (i.e., the ALDH2 gene), and other alcohol-metabolizing enzymes mediate the risk for alcoholism; moreover, these polymorphisms also have an impact on the risk of alcohol-related cancers, such as esophageal cancer. In addition, a gene encoding one of the receptors for the neurotransmitter γ-aminobutyric acid (GABA) known as GABRA2 seems to have a role in the development of alcohol dependence. Genome-wide association studies now offer a host of emerging opportunities, as well as challenges, for discovering the genetic etiology of alcohol dependence and for unveiling new treatment strategies.

o ver the last decade, three large-scale projects have catalyzed a revolution in genetic technologies and studies. First, the Human Genome Project laid the foundation for modern genetic studies of disease by determining the basic sequence of the 3 billion building blocks (i.e., base pairs) that make up the human genome and by identifying the approximately 25,000 genes included in this sequence (www.ornl.gov/sci/ techresources/human_genome/home. shtml). A key component of the Human Genome Project was its collaborative nature, with participating researchers sharing their data within 24 hours of generation. A draft sequence of the human genome was released in 2000 with much fanfare, and the project was completed in 2003 with the release of the final sequence data. Second, the International HapMap Project shortly followed behind as a multicountry effort to identify and catalog common genetic similarities and differences across populations (http://hapmap.ncbi.nlm.nih. gov/). This project built on the knowledge that 99.5 percent of the DNA sequence in humans is identical amongst individuals, and that it is only the remaining 0.5 percent that contribute to the development of diseases and differences in traits. The HapMap project mapped these common differences in the genome and, in 2007, published the human haplotype map with over 3 million identified human genetic variants. Finally, since 2008, the 1000 Genomes Project seeks to discover and more finely catalogue genetic variation, particularly those variants that occur at lower frequencies in different populations (www.1000genomes.org). In addition, this project expands the study of human populations around the world to capture more genetic diversity. The information developed from these three projects has transformed the field of genetics and led to genome-wide association studies (GWASs), which aim to identify regions of the genome that are associated with diseases.
GWASs analyze the presence of hundreds of thousands, or even millions, of polymorphisms across a person's genome. The goal of GWASs is to identify those variants that occur more frequently in people with an illness (e.g., alcoholism) than in those without the disease. Standard GWASs testing now is available, which can query over a million variants that differ in only a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]), indexing variations in the human genome and thereby providing relatively easy access to an individual's genetic makeup (i.e., genotype). Because the cost of genetic testing has decreased with standardization and mass production, GWASs are designed as large-scale studies to examine genetic variation in thousands to tens of thousands of people.

These types of studies already have
Two key functional polymorphisms, developing alcohol dependence is the uncovered thousands of genetic variants both of which are common in Asian same regardless of ethnicity. that alter the risk of developing many populations, have been implicated in Likewise, a variant called rs671 in complex diseases, including type 2 dia-the flushing response to alcohol and the ALDH2 gene results in an amino betes, Crohn's disease, and Parkinson's consequently identified as protective acid change from glutamic acid to disease (Hindorff et al. 2009). Recently, influences on alcohol consumption lysine at position 504 (Glu504Lys) in GWASs also have been applied to the and dependence. In the ADH1B gene, 1 the ALDH enzyme. The allele constudy of alcohol dependence, resulting a polymorphism called rs1229984 taining this variant, called ALDH2*2, in the discovery of additional genes (also referred to as Arg48His), which is associated with substantially reduced that join the existing candidate gene differs from the normal, or wild-type, ALDH activity. As a result of this inacliterature for alcohol dependence. This DNA sequence in a single nucleotide, tivation, acetaldehyde accumulates review will place GWASs in the con-results in an amino acid change at after alcohol consumption and a flushing text of the history of the genetic exam-position 48 in the b subunit of alcohol response occurs, which in turn leads to ination of alcohol dependence.
dehydrogenase from arginine to histia reduced likelihood of alcoholism. For dine (Edenberg 2007). The gene variinstance, Thomasson and colleagues ant (i.e., allele) that encodes histidine (1991) found that only 12 percent of role of Alcohol-metabolizing in place of arginine at amino acid 48 is Chinese men with alcoholism were genes called ADH1B*2; the resulting enzyme ALDH2*2 carriers (i.e., had one or two leads to accelerated oxidation of alcohol copies of the allele), compared with 48 Alcohol dependence was one of the to acetaldehyde and, consequently, percent of nonalcoholic men. Although first disorders to be associated with a increased acetaldehyde accumulation this variant is common in Asian popugenetic contribution. In 1972, facial after alcohol consumption. It has been lations, it rarely occurs in populations flushing and decreased tolerance after linked to reduced alcohol consumpof European and African ancestry. alcohol exposure was observed in sub-tion and a reduced risk of alcoholism. In addition to the widely replicated jects of Asian ancestry (Wolff 1972)-For example, in a recent meta-analysis associations between the rs1229984 a response that is associated with of published studies, Li and colleagues and rs671 with alcoholism risk, certain characteristic alterations in alcohol (2011) reported that the association variants in other genes encoding alcoholmetabolism. Upon ingestion and between rs1229984 and alcoholism is metabolizing enzymes, such as ADH1C, absorption into the blood stream, highly significant (P < 10 -30 ) in Asian ADH4, and ADH5, also have been alcohol first is converted to acetalde-populations. Although the rs1229984 linked to alcohol dependence in other hyde in the liver in a process catalyzed variant is common in populations of populations. Alterations in the ADH1C by the enzyme alcohol dehydrogenase Asian descent, it is found infrequently gene, which encodes the γ subunit of (ADH). Acetaldehyde is a highly toxic (i.e., in less than 5 percent of individualcohol dehydrogenase, can change cancer-inducing substance (i.e., car-als) in populations of European or alcohol metabolism by the encoded cinogen) that normally is converted African descent. Bierut and colleagues protein and have been associated with rapidly to acetate, a less toxic form.
(2012) recently genotyped and deteralcoholism, although these effects are This reaction is mediated by the mito-mined the influence of this variant in not as pronounced as those noted with chondrial enzyme aldehyde dehydroge-three large European and African ADH1B*2 (Edenberg 2007). For nase (ALDH). In this metabolic chain American case-control studies. The instance, the ADH1C variants rs698 of events, two basic mechanisms can results indicated a strong protective and rs1693482 also alter the amino result in the accumulation of acetalde-effect of rs1229984 for alcohol depenacid sequence of the encoded protein hyde in the body-faster metabolism dence (P = 6.6 x 10 -10 ). The allele and occur with high frequency in of alcohol to acetaldehyde, which is also was strongly associated with lower European American populations related to increased ADH activity, alcohol consumption, defined as the (minor allele ≈ 47 percent). Likewise, and/or slower metabolism of acetalde-maximum number of drinks consumed variants in ADH4 and ADH5 sporadihyde to acetate, which is caused by in a 24-hour period (P = 3 x 10 -13 ). cally have been linked to alcoholism. decreased ALDH activity. The excessive Thus, although rs1229984 is rare For example, Macgregor and colleagues production and accumulation of among European and African popula-(2009) reported associations between acetaldehyde then results in the flushing tions, at an individual level the effect SNPs in these genes and alcohol response, which may be accompanied of this ADH1B variant on amount of dependence symptoms as well as quanby lightheadedness, nausea, accelerated alcohol consumed and on the risk of tity, frequency, and maximum drinks, heart rate, and headaches. Because of 1 whereas two other independent studies the unpleasantness of this reaction, dependence (Edenberg et al. 2006;Luo et al. 2006). The study by Mcgregor alcohol dependence and that the samples 4p13 region also contains a cluster of and colleagues (2009) also is among collected to date, although they were genes encoding the α-subunit of the the few studies of non-Asian popula-obtained from thousands of subjects, receptor for the inhibitory neurotranstions that demonstrate an association are not yet sufficient in size to detect mitter γ-aminobutyric acid (GABA), between ADH and ALDH2 polymor-variation in these regions. suggesting that polymorphisms in this phisms and problem drinking.
receptor also may be involved in the In summary, genetic variations in development of alcohol dependence. many of the genes encoding alcohol-

genome-Wide Linkage Studies
This cluster of GABA receptor genes metabolizing enzymes contribute to is of particular interest because of their Genome-wide linkage studies, which differences in alcohol intake and, thus, putative biological relevance to alcoexamine whether large genetic regions the risk for development of alcohol hol-related behaviors, as discussed in distributed across the entire genome dependence. The frequency of these the following section. are cotransmitted along with a disease genetic variants differs dramatically within families, were the first genetic across human populations of Asian, Alcohol Use Disorders and GABRA2 African, and European ancestry. Some Several SNPs in a gene called GABRA2, of these genetic effects on alcohol which encodes the α2-subunit of the metabolism are strongly correlated GABA receptor, have been associated with differences in the risk of developing with alcohol dependence. The first alcohol dependence, whereas others study to implicate GABRA2 in the etihave a more modest effect.
ology of alcoholism was conducted by Edenberg and colleagues (2004), who candidate gene Studies found an association between multiple SNPs (in moderate to high correlation Other early genetic studies of alcohol with each other) and alcohol dependependence relied on candidate gene dence. These results initially were repliassociation studies and genome-wide cated by multiple independent efforts linkage studies. Candidate gene studies (Drgon et al. 2006;Fehr et al. 2003; studies to query the entire genome leveraged the earliest identified genetic Lappalainen et al. 2005; Soyka et al. with hundreds or thousands of genetic variations in specific genes by examining 2008); however, other investigators markers. These studies had two main one gene, and often one variant, at a could not replicate the findings (e.g., characteristics: they were family based, time to determine whether the variant Lind et al. 2008). In contrast to the and the marker coverage across the was associated with alcoholism. These variants in the alcohol-metabolizing genome was modest. Although these experiments identified hundreds of genes genes, the GABRA2 variants that are linkage studies implicated multiple as potentially contributing to alcoholism. associated with alcohol dependence do regions of the genome as being involved For example, in addition to the genes not change the amino acid sequence in in the development of alcoholism, the encoding alcohol-metabolizing enzymes, the encoded protein but instead likely most consistent findings have pointed genes involved in brain signaling (i.e., alter the regulation of GABRA2 protein to two regions on chromosome 4, with neurotransmitter) systems, such as the production. peaks at chromosome locations 4p13dopaminergic, cholinergic, and sero-2 Because the genetic variants do not 4p12 and on 4q21-4q23. The 4q21tonergic systems, have been nominated change the protein structure of the 4q23 region covers the cluster of alcohol for their association with alcohol GABA-α2 subunit, the associations dehydrogenase genes that includes, dependence. In contrast to the alcohol-between these genetic variants and among others, ADH1B, supporting the metabolizing genes, however, these alcoholism are less obvious than those candidate gene findings that alcoholother candidate gene associations have for the alcohol-metabolizing genes. metabolizing genes are associated with not yet been validated in the modern Recent laboratory research has focused alcohol dependence. The chromosome large-scale genetic studies. This lack of on the specific functional aspects of convergence of candidate gene studies GABRA2 in the etiology of alcoholism. and GWASs potentially reflects a large 2 When human chromosomes are prepared for microscopic examination, they take on an x-shaped form, with the shorter arm being GABRA2 encodes a subtype of one of number of false-positive findings from labeled the p arm and the longer one the q arm. The chromo-five subunits that form the commonly the previous candidate gene findings.
somes also are stained and with a certain dye, revealing a characoccurring GABA A ionotropic receptor. Another potential explanation is that teristic pattern of light and dark bands, which helps identify the chromosomes and roughly localize certain chromosome regions.
Binding of GABA to this receptor results, extensive genetic heterogeneity exists, These bands are numbered; accordingly, region 4p13-12 is locat-among several outcomes, in sedation meaning that multiple genes each mod-ed on the short arm of chromosome 4 between bands 12 and 13, and region 4q21-23 is located on the long arm of chromosome 4 and relief of anxiety (i.e., anxiolysis). estly contribute to the development of between bands 21 and 23.
This anxiolytic effect is more closely 276 Alcohol research: C u r r e n t R e v i e w s genetic variations in many of the genes encoding alcohol-metabolizing enzymes contribute to differences in alcohol intake and, thus, the risk for development of alcohol dependence.
related to the α2-subunits than to other ing GABA currents upon ethanol mal models, postmortem brain tissue, subunits (e.g., Dixon et al. 2008).
exposure (Hurley et al. 2009). and novel methodologies that examine Most α2-containing GABA receptors gene expression in the context of neualso are key binding sites for benzodi-• Studies of mice that had been ronal connectivity in alcoholics and azepines, whereas receptors containing genetically modified to produce or nonalcoholics are among a few methods other subunits only show sensitivity not produce the GABA-α2 subunit that may serve to further elucidate the to ethanol. The genetic variants in the (i.e., knock-in and knock-out mice) function of candidate variants identi-  (2011) reported that ognized in candidate gene studies for in the brain's response to preferred P rats show elevated levels of their biological significance in alcoalcoholic drink aromas in a study com-GABA-A subunits, including the holism. The principal disadvantage is paring people who carried two copies α2 subunits, in a brain region called the fairly severe multiple-testing bur-(i.e., were homozygotes) or only one the central nucleus of the amygdala. den (i.e., the need to account for the copy (i.e., were heterozygotes) of the Intriguingly, when molecules that possibility that when 1 million tests "risk" allele for a variant called rs279871.
specifically could inhibit the are conducted, some positive results Finally, Villafuerte and colleagues GABRA2 gene (i.e., α2 silent RNA will be obtained due to chance alone) (2012) reported that two GABRA2 [siRNA]) were introduced into the imposed by GWASs, which results in variants called rs279826 and rs279858, central amygdala, even the P ani-the requirement for statistical signifiwhich previously had been implicated mals no longer demonstrated binge cance to be denoted by P values of in the etiology of alcoholism, not only drinking for a short period of time, 5 x 10 -8 and lower. This represents a correlated with impulsiveness but also and this effect correlated with a sig-very high significance level for validawith activity in a brain region called nificant reduction of α2 levels. This tion; consequently, many SNPs that the insula cortex during monetary effect did not occur when siRNA truly are associated with alcoholism reward and loss. Thus, in experiments for another receptor subunit (i.e., risk may not yet have been recognized assessing responses during reward α1 siRNAs) was used or when α2 because they have not yet surpassed anticipation and loss, study participants siRNA was introduced in other this threshold. from alcoholic families with the haplocontrol regions of the brain. Four GWASs of alcohol dependence type that is associated with increased and two of alcohol consumption now alcoholism vulnerability showed signif-Thus, much research has examined have been completed in populations of icantly higher activation in the left the role of the α-subunit of the GABA European ancestry. In the first of these, insula than did participants with a receptor, and this protein clearly plays Treutlein and colleagues (2009) found different haplotype. However, much a central role in alcohol-mediated anxiseveral SNPs with P values in the range more research is needed to understand olysis and other effects of alcohol use. of 10 -6 ; upon pooling with a replication the biology underlying these genetic Nevertheless, it remains important to sample, two of these SNPs, rs7590720 associations with GABRA2. more clearly connect genetic variations and rs1344694, showed statistical sig-Animal models also have begun to found to be associated with alcoholism nificance in the combined male-only address these functional aspects of risk in humans with basic biologic sample of 1,460 alcohol-dependent GABRA2, as follows: function. Functional studies using anisubject and 2,332 community control subjects. These SNPs are located in a • Work with frog eggs (i.e., Xenopus 3 The investigators assessed the blood-oxygen-level dependence region of the long arm of chromosome oocytes) has demonstrated that the (BoLD) response, which represents a measure of blood flow through and thus, indirectly, brain activity using a technique called 2 where prior linkage studies have α2 subunits are involved in regulatfunctional magnetic resonance imaging (fMRI).
identified increased allele-sharing for alcoholism. The gene closest to the sponding 5-fold decreased likelihood month period when the participants association signal, PECR (which encodes of developing alcohol problems consistent indicated that they drank the heaviest. the enzyme peroxisomal trans-2-enoyl-with alcohol dependence. Because this The results implicated variants in the CoA reductase) is involved in the variant is rare in subjects of European TMEM108 gene, which encodes a metabolism of fatty acids, particularly and African descent, this strong finding transmembrane protein of unknown during deprivation when energy is not seen, nor expected, in non-Asian function (P = 1.2 x 10 -7 ). These two expenditure transitions from carbohy-populations. initial studies of alcohol consumption drates to fatty acids. In addition, the In addition to studying alcohol implicate different genomic regions, investigators noted that a SNP called dependence, a strategy to increase the and their findings have not converged. rs1614972 in the ADH1C gene was power of GWASs has been to examine With larger sample sizes, it will strongly associated with alcohol depen-alcohol consumption as a quantitative become clearer whether these results dence (P = 1.4 x 10 -4 ) even though variable. A recent meta-analysis of alcohol represent true findings. it did not meet strict standards for consumption by Schumann and genome-wide multiple testing. Two colleagues (2011)  . Across the four consumption was 11 liters in Russia studies, there was no evidence for repliand 8.5 liters in the United States, cation of any genetic signals, raising with much lower consumption levels the possibility that the identified signals based subjects and a follow-up sample reported in parts of Asia and northern were false positives or that true signals of 21,185 European American subjects Africa. Although modest alcohol concould not be detected as a result of the to identify a SNP called rs6943555 in sumption is associated with health (by GWASs standards) relatively small samthe autism susceptibility candidate 2 (e.g., cardiovascular) benefits, accordple sizes (i.e., several thousand subjects). (AUTS2) gene that was associated with ing to the WHO, 2.5 million people Although GWASs of subjects of alcohol consumption (in grams/day/ die each year from the harmful effects European descent have struggled to kilogram of body weight) at P < 5 x of alcohol (http://www.who.int/subidentify and replicate SNPs associated 10 -9 . Specifically, people who carried stance_abuse/facts/en/). In the United with alcohol dependence, a recent the minor allele of rs6943555 showed States, nearly 80,000 people die annu-GWASs of a sample of 1,721 Korean 5.5 percent lower alcohol consumption ally from the short-and long-term men reported associations with P values than people who carried the major consequences of alcohol use (Mokdad of 10 -58 and lower for SNPs in moderate allele; however, secondary analyses did et al. 2004). Excessive alcohol conto high linkage disequilibrium 4 with not reveal any association with alcohol sumption can lead to alcohol depen-SNPs in the ALDH2 gene, including dependence. Follow-up studies showed dence, which affects 12.5 percent of rs671 (Baik et al. 2011). In this study, changes in mRNA expression that cor-people in the United States across their carriers of the protective allele of the related with the rs6943555 genotype; lifetime (Hasin et al. 2007). Thus, the SNP rs11066280 in the gene C12orf51, moreover, the gene itself was expressed health effects of alcohol consumption which frequently occurs together with differently in low-alcohol-versus high-remain a public health priority that rs671 in ALDH2 (the SNP that inactialcohol-preferring rats. Likewise, needs to be studied from all angles, vates ALDH2), had a 13-fold increased Heath and colleagues (2011) examined including improving prevention and risk for alcohol flushing and a correa quantitative factor score created from treatment, while also examining basic items assessing quantity and frequency biological underpinnings. of consumption, frequency of intoxica-Alcohol consumption increases the tion, and maximum drinks in a single likelihood of several other disorders. 24-hour period, all reported for the 12-For example, population-based studies 278 Alcohol research: C u r r e n t R e v i e w s The health effects of alcohol consumption remain a public health priority that needs to be studied from all angles, including improving prevention and treatment, while also examining basic biological underpinnings.
have demonstrated that esophageal percent of European populations. mechanisms is to gain new insights cancer is linked to alcohol consumption Nevertheless, it is by far the strongest into potential treatment approaches. as a result of the tissue's exposure to genetic variant that alters the risk for To some extent, clinicians already ethanol and its metabolite acetaldehyde. the development of UADT cancer, are exploiting the understanding of Consequently, functional variants in even after accounting for alcohol con-genetic variation in the development of ADH1B and ALDH2 that affect alcohol sumption. Variants in other alcohol-alcoholism in their treatment strategies. clearance and acetaldehyde accumulation metabolizing genes also have been For example, the variation in enzyalso play a role in the development of implicated in the development of matic pathways of alcohol metabolism esophageal cancer. The effects of these esophageal cancer. found in the population is mimicked variants on cancer are strongly moderated in the pharmacologic treatment of In several of these studies, the effects by accompanying alcohol exposure.
alcohol dependence using the medicaof ADH1B and ALDH2 variants pre-For example, people who carry the tion disulfiram, which inhibits ALDH dominantly were noted in those who ADH1B*2 allele that results in more and is a pharmacologic treatment simidrink alcohol. For instance, even in rapid ethanol conversion to acetaldehyde, lar to the natural variation related to people with two copies of the ALDH2*2 experience flushing and, hence, have rs671 and decreased ALDH activity allele, which is highly protective for reduced likelihood of alcohol intake, in Asians. If alcohol is consumed after alcohol intake, the few drinkers are at a protecting them from esophageal taking disulfiram, acetaldehyde levels profoundly elevated risk for esophageal cancer. Conversely, people who carry increase, causing flushing, nausea, cancer (Yang et al. 2010). It might be the ADH1B*1 variant, which results vomiting, and headache in the patient, hypothesized that this finding reflects in slower alcohol metabolism, are at which produces an aversive response to a genotype-environment correlation increased risk for esophageal cancer, continued alcohol use. where ALDH2 and ADH1B variants arguably because of extended tissue influence the likelihood of continued exposure to ethanol. Likewise, having alcohol (and acetaldehyde) exposure, the ALDH2*2 allele, which inactivates

Future Directions
which, in turn, serves as the environaldehyde dehydrogenase, has protective mental risk factor for the development effects on both alcohol intake and Researchers long have known that of esophageal cancer, either as a result esophageal cancer. However, people alcohol dependence clusters in families. of prolonged ethanol exposure or of who carry only one copy of ALDH2*2 During the past 50 years, adoption and the carcinogenic effects of acetaldehyde. often continue to drink, which leads to twin studies definitively have demon-An alternative explanation is that of a acetaldehyde accumulation and consestrated that this clustering is related quently increases the risk for esophageal genotype x environment interactioncancer (Brooks et al. 2009). that is, people who carry the ALDH2 Because both protective gene variants and ADH1B genotypes that protect are more common in Asian populations, against alcohol intake might be exquisitely several studies have demonstrated the sensitive to the joint effects of genoinfluence of ADH1B*2 and ALDH2*2 type and alcohol consumption on their on esophageal cancer in these popula-vulnerability to esophageal cancer. tions (Ding et al. 2010;Hashibe et al. 2008). A meta-analysis of published studies on Asian populations (Yang et  level with the identification of addi-SNPs in determining the diagnosis, the explained genetic variance to date tional genes that contribute to alco-prognosis, and treatment of addiction. is less than 1 percent, which raises the holism. Several challenges remain, Dr. Bierut served as a consultant for question "Where is the missing genetic however, as this field moves forward. Pfizer in 2008. No other potential variance?" Three main explanations First, investigators must increase the conflicts of interest relevant to this have been given for the missing variance diversity of the populations under study. article were reported. in the genetic risk for alcohol depen-Varying allele frequencies across popudence: (1) many genes of small effect lations mean that important genetic may be involved that do not yet surpass contributors in one population may references the stringent threshold for genomenot be seen in another, with the classic wide significance; (2) rare variationsexample being the variation in the under study will allow researchers to wide association study of alcohol dependence.
genotyping; and (3) other mechanisms, leverage these differences to refine asso-Proceedings of the National Academy of Sciences of the including the interplay of genes and ciation signals and also to increase the United States of America 107 (11):5082-5087, 2010.
environments, may contribute that are potential discovery of genetic variants. two methods only have converged that permit the study of large numbers modestly, primarily with the alcohol-